CN1185181A - Method and amplifying specific nucleic acid squences - Google Patents
Method and amplifying specific nucleic acid squences Download PDFInfo
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- CN1185181A CN1185181A CN96194151A CN96194151A CN1185181A CN 1185181 A CN1185181 A CN 1185181A CN 96194151 A CN96194151 A CN 96194151A CN 96194151 A CN96194151 A CN 96194151A CN 1185181 A CN1185181 A CN 1185181A
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Abstract
The present invention provides a method of detecting a genetic polymorphism in an individual. In one form, the method comprises the following steps: (1) obtaining a sample containing a nucleic acid from the individual; (2) amplifying the nucleic acid sample from step (1) by a process involving thermocycling and primers, the amplification occurring in the presence of a thermostable restriction endonuclease which retains activity during thermocycling, the primers being selected such that they introduce a sequence recognized by the thermostable restriction endonuclease into the nucleic acid amplified from nucleic acid either not including the polymorphism or including the polymorphism; and (3) analyzing the product of step (2) to determine the presence of the polymorphism.
Description
Invention field
The present invention relates to the method for amplification in vitro specific nucleic acid target sequence.Particularly the present invention relates to the method with thermotolerance restriction endonuclease mediation selective amplification nucleic acid target sequence, this target sequence contains sequence difference, comprises point mutation, disappearance and insertion.
Background of invention
Multiple genetic diseases is relevant with heritable variation such as point mutation, disappearance and insertion with acquired disease.Some is directly related with the disease appearance in these variations, and other is then relevant with disease risks and/or prognosis.Owing to single gene mutation causes having 500 kinds of human inheritance's diseases, these comprise cystic fibrosis, muscular dystrophy, alpha1-antitrypsin defective, phenylketonuria, Dresbach's anemia or proterties and various other hemoglobinopathy.In addition, the individuality to several frequently seen polygene state such as atherosclerosis heart trouble have the susceptibility of increase has demonstrated relevant with the heredity of specific dna sequence polymorphism.Cancer is thought owing to relate to due to the damage accumulation of cell proliferation or differentiation gene.Ras proto-oncogene K-ras, N-ras and H-ras and p53 tumor suppressor gene are the gene examples of frequent sudden change in human cancer, and special sudden change causes the conversion capability that activates or increase in these genes.Genetic analysis becomes conventional clinically analysis probably, is used to evaluate disease risks, medical diagnosis on disease, and prediction patient's prognosis or reaction to treating, and be used to monitor PD.The importing of this heredity test depends on simple, the cheap and Genetic Variation Analysis method fast of exploitation.
In situation seldom,, sudden change can detect if suddenling change when by chance being positioned at natural restriction endonuclease identification/cleavage site.WO84/01389 has described the method whether a kind of existence by screening restriction endonuclease site distinguishes wild type gene and non-wild-type muton.The contriver for example understands this principle by the varient sequence of analyzing human H-ras proto-oncogene codon 12 positions.Wild-type sequence codon 12 has formed the part of restriction endonuclease NaeI and HpaII identification/cleavage site.Can distinguish the wild-type proto-oncogene and have the activated oncogene of sudden change at this codon place with these endonuclease enzymic digestions.The point mutation of H-ras codon 12 frequently is found in the basis that bladder cancer and this strategy can form medical diagnosis screening reagent box.
The nucleic acid amplification in vitro method has been widely used in genetics and medical diagnosis on disease.Polymerase chain reaction (PCR) is the segmental sensitive method (R.K.Saiki etc. of strong amplification in vitro specific nucleic acid, 1985, science 230,1350-1354 and F.F.Cheheb etc., 1987, nature 329,293-294 and US4683202, US4683195, US4800159, US4965188 and US5176995).PCR is mediated by Oligonucleolide primers, and primer is positioned at the flank of target sequence to be synthesized, and is complementary to the sequence that is positioned at the template DNA opposite strand.These steps in reaction owing to temperature cycle (thermal cycling) takes place.Template DNA allows primer and target sequence anneal at first through heat denatured thereby will react cooling then, and primer is extended by archaeal dna polymerase then.Sex change, annealing and the circulation of DNA synthetic repeat repeatedly and every template of taking turns the product of amplification as next round.This process causes the index amplification of amplicon, and it does not hold the new synthetic sequence of mixing Oligonucleolide primers and containing between primer to copy 5 '.
PCR uses extremely extensively and has developed the improved form of many this basic skills.The primer that is used for PCR can match fully with target sequence or they can contain the base of mispairing or modification.The additional sequences of primer 5 ' end can be beneficial to be obtained pcr amplification and comprises that the primer of mark can be beneficial to detection.Comprise that in primer into mismatched bases can cause producing new restriction endonuclease identification/cleavage site, these sites can be positioned at primer sequence fully, perhaps, they can cross over sequence (J.B. and the A.D.Levinson (1988) that part is positioned at primer and partly is positioned at new synthetic target sequence, nature 334,119-124).The general rule that design contains nearly 3 ' end base mismatch primer set up (S.Kwok etc. (1990), nucleic acids research 18,999-10005).
The Mdification primer that contains base mismatch is used for to H-ras amplicon (R.Kumar and M.Barbacid (1988), oncogene 3,647-651) the middle identification/cleavage site that imports new restriction endonuclease in codon 12 sudden changes.Similarly, the primer that contains base mismatch is used to be called allele-specific enrichment (leukemia such as Todd AV, 1991,5:160) or enrichment PCR (Levi S etc., cancer research, 1991, in method 6:1079), these are the very sensitive methods of check point sudden change.In these methods, are wild-types as infructescence at codon 12, the DNA sample is with importing EcoNI site in the N-ras amplicon, or the primer in importing BstNI site increases in the K-ras amplicon.Thereby the sample aliquot of PCR reaction solution is with suitable restriction endonuclease digestion cutting wild-type amplification before second takes turns the anti-amplicon that digests that increases again in the PCR circulation.These methods cause preferentially increasing codon 12 in ras has the sequence of point mutation.Recently, a kind of simplification enrichment PCR method (Singh etc., international cancer magazine, 1994 of carrying out in the single pipe that allow to be reflected at are disclosed; 5:1009), this method also needs to take turns initial pcr amplification, yet restriction endonuclease directly joins in the reaction tubes then.After restriction nuclease enzyme incubation, second takes turns PCR circulation causes increasing have the sequence of sudden change in restriction endonuclease identification/cleavage site.Natural or the inductive limiting acid endo enzyme site needs dna polymerase activity that is used for PCR subsequently then is the activity that is used for the restriction endonuclease of cutting analysis in this analysis pcr amplification.The enrichment PCR method at first needs to be used for the dna polymerase activity of PCR, be then the restriction endonuclease activity to cut special sequence, then further be dna polymerase activity with the amplicon of the anti-digestion of amplification again.
In the PCR process, utilize the active ability of restricted ribozyme restriction endonuclease and archaeal dna polymerase that the advantage of several respects may be provided simultaneously, it can allow to be formed for containing all reagent when PCR is initial, comprises the simple method of the varient sequence that increases separately or preferentially in the reaction of enzyme.Do not know in the past whether in PCR, to comprise that restriction endonuclease into can cause (i) varient that complete (or part) suppresses and (or preferential) amplification lacks the sequence of restriction system property endonuclease identification/cleavage site (ii) separately to the sequence amplification that contains restriction endonuclease identification/cleavage site.Suppress sequence amplification fully and/or the ability of the varient sequence that increases separately can cause forming and need not before analyzing further method of operating.The number of steps minimizing of required selective amplification and/or subsequent analysis amplicon can cause forming faster, less labour intensity and/or being easier to automated method.Further advantage is to react and will finishes in closed system and this will reduce the chance of polluting in the PCR process.
This method will simultaneously need be in the restriction endonuclease under the condition compatible with PCR and the activity of archaeal dna polymerase.The essential i of this restriction endonuclease and archaeal dna polymerase) in same reaction conditions that must be compatible with PCR (for example, salt, PH) the performance function and ii) must be in these reaction conditionss enough underground heat stable with PCR need working cycle in keep activity.Be suitable for the temperature must be in the PCR process compatible, have activity when typically being 50 ℃-65 ℃ with the stringent condition of primer annealing with the restriction endonuclease of PCR associating.Activity had been developed the amplification in vitro (EPO384315AI) that is used for mediating the isothermal reaction that is called the alternative amplification of chain in the past in the time of hot resistant DNA polymerase and restriction endonuclease.Do not know in the past whether restriction endonuclease can have enough thermostabilitys to keep activity in the required thermal cycling process of PCR.
Summary of the invention
In first aspect, the present invention includes a kind of method that detects genetic polymorphism in the individuality, this method comprises that the following step poly-:
(1) obtains the sample that contains individual amplifying nucleic acid;
(2) nucleic acid samples of the method amplification step (1) by comprising thermal cycling and primer, amplification is to keep in the presence of the active heat resistant restriction ribozyme restriction endonuclease to carry out in the thermal cycling process, selects primer so that they can import the sequence of being discerned by heat-stable restriction endonuclease from nucleic acid that does not comprise polymorphism or amplification in amplification in the nucleic acid of the nucleic acid that comprises polymorphism; With
(3) product of analytical procedure (2) is to detect whether existing of polymorphism.
Primer will be imported amplification in the nucleic acid of the nucleic acid that will not comprise polymorphism by the sequence of heat resistant restriction endonuclease identification in the embodiment of the present invention aspect this.
Aspect second, the present invention includes a kind of method that detects genetic polymorphism in the individuality, this method comprises following step:
(1) obtains the sample that contains individual nucleic acid;
(2) nucleic acid samples of the method amplification step (1) by comprising thermal cycling and primer, amplification is to have active heat resistant restriction endonuclease at the same time to exist down and carry out, and the selection of restriction endonuclease is to make its identification not comprise the nucleic acid of polymorphism and nonrecognition comprises the nucleic acid of polymorphism or their are discerned comprises the nucleic acid of polymorphism and nonrecognition does not comprise the nucleic acid of polymorphism; With
(3) product of analytical procedure (2) is to detect whether existing of polymorphism.
The identification of heat resistant restriction endonuclease does not comprise the nucleic acid of polymorphism in the embodiment of the present invention aspect this.
Additional step below this method further comprises in the preferred embodiment of the present invention:
(4) with the nucleic acid and the reaction of at least a restriction endonuclease of amplification in the step (2), the selection of this at least a restriction endonuclease is to make its digestion comprise the amplification of nucleic acid of specific polymorphism; With
Whether (5) detect step (4) digestion and take place, digestion is the sign that specific polymorphism exists.
Many technology that are used for amplification of nucleic acid that comprise thermal cycling are arranged, and these technology comprise polymerase chain reaction (PCR), and ligase chain reaction (LCR) is based on amplification of transcribing and restrictive amplification.Yet the method that preferably includes thermal cycling now is PCR.
The analysis of step in another embodiment preferred (3) comprises that whether the nucleic acid that detects step (2) amplification exists, the nucleic acid of amplification whether exist represent polymorphism existence whether.
Method of the present invention can be used for various types of nucleic acid simultaneously, and typically nucleic acid is DNA.
The heat resistant restriction endonuclease is selected from BstNI, BslI, Tru9I and Tsp 509I in another embodiment preferred of the present invention.
The inventive method can be used for detecting the genetic polymorphism of certain limit, comprise and come across following one of them polymorphism: ras proto-oncogene such as K-ras, N-ras and H-ras, or p53 tumor suppressor gene, or HIV-I, film conduction regulon, alpha antitrypsin or beta-globin are changeed in cystic fibrosis.The inventive method is particularly useful in the polymorphism that detects K-ras codon 12 positions.
The inventive method can be used for analyzing the genetic polymorphism of certain limit, comprises point mutation, little disappearance and insertion.Found that the heat resistant restriction endonuclease can be enough heat-resisting to keep activity in the thermal cycling process, found that also PCR can carry out keeping under the active and stable on heating same buffer condition of restriction endonuclease with various polysaccharases.Found in the PCR process, to comprise that into the thermotolerance restriction endonuclease can cause (i) to suppress the varient that contains the amplification of restriction endonuclease identification/cleavage site sequence and (ii) only increase disappearance restriction endonuclease identification/cleavage site sequence.These find that permission forms the method for the selectivity PCR (REMS-PCR) of restriction endonuclease mediation.REMS-PCR is more simple than other PCR method, and it utilizes the variation of restriction endonuclease analytical sequence.All reacted constituents just exist when PCR is initial and operation that need not be subsequently before analyzing, and therefore reaction can be finished in airtight container or cell.Found also in the PCR process, to comprise that part that heat resistant restriction endonuclease into can cause (i) to contain the nucleic acid amplification of restriction endonuclease identification, cleavage site suppresses and the varient of (ii) preferential amplification disappearance restriction endonuclease identification/cleavage point range.
Detailed description of the present invention
As used herein, following term and phrase are defined as follows:
PCR is the external DNA cloning method of 2 of a kind of needs primer of being positioned at target sequence to be synthesized both sides.Primer is one section oligonucleotide sequence, and it can hybridize on the target sequence and in the PCR process in sequence-specific mode and extend.Amplicon (Amplicons) or PCR product or PCR fragment are the extension products that comprises primer and new synthetic target sequence copy.The multiplex PCR system contains many cover primers, and it causes producing simultaneously a plurality of amplicons.Primer can mate fully with target sequence or they can contain inner mismatched bases, and this base can cause importing restriction nuclease enzyme identification/cleavage site in special target sequence.Primer also can contain the Nucleotide of extra sequence and/or modification or mark so that obtain or detect amplicon.The annealing of the thermally denature of DNA, primer and its complementary sequence and annealing primer cause the index amplification of target sequence with the recirculation of the extension of archaeal dna polymerase.Term target or target sequence refer to nucleotide sequence to be amplified.The term template refers to original nucleic acid to be amplified.
The selectivity PCR (REMS-PCR) of restriction endonuclease mediation uses a kind of analytical procedure that the inventive method is set up by the inventor.This method simultaneously need be in the PCR process restricted property endonuclease and dna polymerase activity.The restriction endonuclease that is suitable for REMS-PCR preferably with Oligonucleolide primers compatible temperature of annealed stringent condition in the PCR process, have activity when typically being 50 ℃-65 ℃.In this scope, have one group of commercial available restriction endonuclease that high thermophilic educates temperature and be listed in the table below 1.
Term " individuality " this paper uses and is intended to include human and non-human animal, bacterium, yeast, fungi and virus with broad sense.
Table 1
The A=VITAMIN B4, G=guanine, T=thymus pyrimidine, C=cytosine(Cyt), N=A or G or T or C
| Restriction enzyme | Identification/cutting sequence | Best heated culture temperature |
| Acc?III | ?TCCGGA | ?65℃ |
| AcsI/ApoI | ?A/G)AATT(T/C) | ?50℃ |
| Acy?I | ?G(A/G)CG(C/T)C | ?50℃ |
| Bco?I | ?C(C/T)CG(A/G)G | ?65℃ |
| Bsa?BI/Bsi?BI | ?GATNNNNATC | ?60℃/55℃ |
| Bsa?MI | ?GAATGCN | ?65℃ |
| Bsa?II | ?CCNNGG | ?60℃ |
| Bsa?OI | ?CG(A/G)(T/C)CG | ?50℃ |
| Bsa?WI | ?(A/T)CCGG(A/T) | ?60℃ |
| Bsc?BI | ?GGNNCC | ?55℃ |
| Bsc?CI | ?GAATGCN | ?65℃ |
| Bsc?FI | ?GATC | ?55℃ |
| Bse?AI | ?TCCGGA | ?55℃ |
| Bsi?CI | ?TTCGAA | ?60℃ |
| Bsi?EI | ?CG(A/G)(C/T)CG | ?55℃ |
| Bsi?HKAI | ?G(A/T)GC(A/T)C | ?65℃ |
| Bsi?LI | ?CC(A/T)GG | ?60℃ |
| Bsi?MI | ?TCCGGA | ?60℃ |
| Bsi?QI | ?TGATCA | ?60℃ |
| Bsi?WI | ?CGTACG | ?55℃ |
| Bsi?XI | ?ATCGAT | ?65℃ |
| Bsi?ZI | ?GGNCC | ?60℃ |
| Bs/I | ?CCNNNNNNNGG | ?55℃ |
| Bsm?I | ?GAATGCN | ?65℃ |
| Bsm?AI | ?GTCTCN 1/N 5 | ?55℃ |
| Bsm?BI | ?CGTCTCN 1/N 5 | ?55℃ |
| Bss?TII | ?CC(A/T)(A/T)GG | ?50℃ |
| Bst?I | ?ACTGGN | ?65℃ |
| Bsr?DI | ?GCAATGNN | ?60℃ |
| Bst?TII | ?GCAGCN 8 | ?50℃ |
| Bst?BI | ?TTCGAA | ?65℃ |
| Bst?NI | ?CC(A/T)GG | ?60℃ |
| Bst?UI | ?CGCG | ?60℃ |
| Bst?YI | ?(A/G)GATC(C/T) | ?60℃ |
| Bst?ZI | ?CGGCCG | ?50℃ |
| Dsa?I | ?CC(A/G)(C/T)GG | ?55℃ |
| Mae?II | ?ACGT | ?55℃ |
| Mae?III | ?GTNAC | ?55℃ |
| Mwo?I | ?GCNNNNNNNGC | ?60℃ |
| Ssp?BI | ?TGTACA | ?50℃ |
| Taq?I | ?TCGA | ?65℃ |
| Tfi?I | ?GA(A/T)TC | ?65℃ |
| Tru?9I | ?TTAA | ?65℃ |
| Tsp?45I | ?GT(C/G)AC | ?65℃ |
| Tsp?509I | ?AATT | ?65℃ |
| Tsp?RI | ?NNCAGTGNN | ?65℃ |
| Tth?IIII | ?GACNNNGTC | ?65℃ |
In order more clearly to understand character of the present invention, its preferred form is described with reference to the following examples.
Embodiment 1
Estimate restriction endonuclease activity/stable on heating test
Activity/thermal test is used for after the thermal cycling of certain number detecting the restricted sour restriction endonuclease that comprises BstNI, BslI, Tru9I and Tsp 509I thermotolerance and the remaining enzymic activity at various Laemmli buffer system Laemmlis.
Compare BstNI, BslI and the activity/thermotolerance of Tru9I in multiple buffer conditions.In the total reaction volume of 25 μ l, contain primer (as shown in table 2 below), each 100 μ M of dNTP (dATP, dCTP, dTTP, dGTP), the Taq archaeal dna polymerase of 0.5 unit (5 units/μ l; AmpliTaq, Perkinelmer Inc.), and or BstNI (10 units/μ l of 20 units; New England Biolabs) or Tru9I (10 units/μ l; BoehringerMannheim) or the BslI of 10 units (50 units/μ l; New England Biolabs).Table 2
| Primer | Consumption (pmole) | In analyzing, following enzyme exists | Sequence |
| 5BKIT | 7.5 | BstNI | TATAAACTTGTGGTAGTTGGACCT |
| 5BKIQ | 7.5 | BslI，Tru?9I | TATAAACTTGTGGTACCTGGAGC |
| 3KiE | 7.5 | BstNI，Bsl?I， Tru?9I | CTCATGAAAATGGTCAGAGAAACC |
| 5BKIW | 1.25 | BslI | TTTTGTCGACGAATATGATCC |
In addition, reaction contains one of following ealkaline buffer system (listing in table 3), has or do not have various additional agents.
Table 3
| The ealkaline buffer title ***New England Biolabs **Boehringer Mannheim *Perkin?Elmer | Salt | Tris Hcl (pH in the time of 25 ℃) | MgCl 2mM | DTT mM |
| ***NEB2 **SuRE/CutM | 50mM?NaCl | 10mM(7.9) | 10 | 1 |
| ***NEB3 | 100mM?NaCl | 50mM(7.9) | 10 | 1 |
| *PCR damping fluid II | 50mM?KCl | 10mM(8.3) | ||
| *The Stoffel damping fluid | 10mM?KCl | 10mM(8.3) | ||
| MTris?10 | 50mM?NaCl | 10mM (8.0 or 8.3 or 8.5 or 8.75) | ||
| Htris?50 | 100mM?NaCl | 50mM (8.0 or 8.3 or 8.5 or 8.75 or 9.0 or 9.5) |
Reaction places GeneAmp PCR system 9600 thermal cyclers (perkin elmer), is heated to high temperature and by thermal cycling shown in the table 4.
Table 4
| Restriction endonuclease | BstNI | BslI | Tru9I |
| Starting temperature | 94 ℃ 2 minutes | 92 ℃ 1 minute | 94 ℃ 2 minutes |
| Thermal cycling | 60 ℃ 1 minute 92 ℃ 20 seconds | 55 ℃ 1 minute 92 ℃ 20 seconds | 65 ℃ 1 minute 92 ℃ 20 seconds |
| The thermal cycling number | 15 or 30 | 15 or 30 | 15 or 30 |
| Thermophilic is educated temperature | 60℃ | 55℃ | 65℃ |
After the thermal cycling, with 8 μ g plasmid DNA (pGFP-Cl in the 5 μ l volumes; Clontech) join in every pipe and be reflected at the optimum temps that manufacturers lists and carried out 1 hour.The ability of restriction endonuclease cutting plasmid DNA is by (FMC Bioproducts, Rockland MD) go up electrophoresis and measured at the 3%NusieveGTG gel.Endonuclease is divided into inactivation (I); Have low (L), medium (M) or high (H) activity; Or have fully (F) active (table 5).
Table 5
| Ealkaline buffer | Extra reagent | Bst NI activity | The BslI activity | Tru 9I activity | |||
| Cycle number | Cycle number | Cycle number | |||||
| ??15 | ??30 | ??15 | ??30 | ??15 | ??30 | ||
| ??NEB2 ??SuRE?Cut?M | ??M | ??L | ???M | ??L | |||
| ??NEB3 | ??F | ??M | |||||
| 1 * PCR damping fluid II | ??3?mM?MgCl 2 | ??M | ??L | ||||
| ??6?mM?MgCl 2 | ??M | ??L | ??I | ??I | |||
| ??10?mM?MgCl 2 | ??H | ??M | ??I | ??I | |||
| ??10?mM?MgCl 2·1mM?DTT | ??H | ??M | |||||
| 1 * Stoffel damping fluid | ??3?mM?MgCl 2 | ??M | ??L | ||||
| ??6?mM?MgCl 2 | ??M | ??L | ??I | ??I | |||
| ??10?mM?MgCl 2 | ??M | ??L | ??I | ??I | |||
| ??10?mM?MgCl 2·1mM?DTT | ??H | ??M | |||||
| ??MTris?10?pH?8.3 | ??10?mM?MgCl 2 | ??H | ??L | ??I | ??I | ||
| ??HTris?50?pH?8.3 | ??F | ??M | |||||
| ??MTris?10?pH?8.0 | ??10?mM?MgCl 2·1mM?DTT | ||||||
| ??MTris?10?pH?8.3 | ??10?mM?MgCl 2·1mM?DTT | ??H | ??M | ||||
| ??MTris?10?pH?8.0 | ??10?mM?MgCl 2 | ??M | ??L | ||||
| ??MTris?10?pH?8.3 | ??H | ??L | ??I | ??I | |||
| ??MTris?10?pH?8.5 | ??M | ??L | ??I | ??I | |||
| ??MTris?10?pH?8.75 | ??I | ??I | |||||
| ??10?mM?MgCl 2·1mM?DTT | ??H | ??M | ???H | ??L | |||
| ??HTris?50?pH?8.5 ??HTris?50?pH?8.5 | ??6?mM?MgCl 2 | ??I | ??I | ||||
| ??6?mM?MgCl 2·1mM?DTT | ??H | ??M | |||||
| ??HTris?50?pH?8.0 | ??10?mM?MgCl 2 | ??F | ??M | ||||
| ??HTris?50?pH?8.3 | ??F | ??M | ??I | ??I | |||
| ??HTris?50?pH?8.5 ??HTris?50?pH?8.5 | ??F | ??M | ??I | ??I | |||
| ??10?mM?MgCl 2·1mM?DTT | ??H | ??M | ???H | ??L | |||
| ??HTris?50?pH?8.75 | ??10?mM?MgCl 2 | ??I | ??I | ||||
| ??10?mM?MgCl 2·1mM?DTT | ??H | ??M | ???H | ??L | |||
| ??HTris?50?pH?8.3 | ??3?mM?MgCl 2 | ??H | ??M | ||||
| ??6?mM?MgCl 2 | ??F | ??M | |||||
| ??10?mM?MgCl 2 | ??F | ??M | ??I | ??I | |||
| ??HTris?50?pH?8.3 | ??10?mM?MgCl 2·1mM?DTT | ??H | ??M | ||||
| ??HTris?50?pH?8.3 ??6mMMgCl 2 | ??- | ??F | ??M | ||||
| ??1?mM?DTT | ??H | ??M | |||||
| 0.1 mg/ml acetylize BSA (aBSA) | ??H | ??M | |||||
| 0.1 the non-acetylize BSA of mg/ml (non-a BSA) | ??H | ??M | |||||
| ??1?mMDTT+aBSA | ??M | ??M | |||||
| ??1?mM?DTT++non-aBSA | ??M | ??L | |||||
| 10% glycerine | ??H | ??M | |||||
| ??HTris?550?pH?9.0 | ??10mMMgCl 2·1mM?DTT | ??H | ??M | ||||
| ??HTris?50?pH?9.5 | ??10mMMgCl 2·1mM?DTT | ??F | ??M | ||||
These experiments show that the activity/thermotolerance of restriction endonuclease in the thermal cycling process is very different according to restriction endonuclease and its residing Laemmli buffer system Laemmli.PH in the Tris damping fluid and ionic strength, univalent cation (K
+Or Na
+) selection and concentration, free Mg
2+Concentration and the existence of existence, the especially DTT of other additive can influence activity/thermotolerance.The influence of every kind of composition can depend on other composition in the damping fluid in these compositions, and for example, BstNI is containing 10mM MgCl
2PCR damping fluid II in contain 3 or 6mMMgCl
2Damping fluid in keep more active.On the contrary, between 3～10mM, change MgCl when in HTris 50 (pH8.3) damping fluid
2Concentration to the almost not influence of Bst NI activity.In another example, pH of buffer has bigger influence to Bst NI thermostability/activity at MTris 10 in than HTris 50.
Bst NI is through keeping complete activity and keep medium activity after 30 thermal cyclings in the Laemmli buffer system Laemmli one of below containing after 15 thermal cyclings: i) 100mM NaCl, 50mMTrisHCl (pH8.3) and 6mM MgCl
2Or ii) 100mM NaCl, 50mM TrisHCl (pH8.0-8.5) and 10mM MgCl
2Or iii) NEB3 damping fluid and 0.1mg/ml.Bst NI has in these damping fluids in the NEB2 damping fluid of 0.1mg/ml acetylize BSA of buffer conditions of manufacturer recommendation and has more high reactivity in the thermal cycling process.
Similar detection Bsl I activity/the thermostability experiment shows in order to keep this endonuclease activity after thermal cycling needs the existence of 1mM DTT.If DTT exists, BslI is retentive activity in condition and range widely.Keep medium activity after taking turns thermal cycling through 30 in the Laemmli buffer system Laemmli of BslI below containing: i) PCR damping fluid II (perkin elmer), 1mM DTT and 10mM MgCl
2, ii) Stoffel damping fluid (perkin elmer), 1mM DTT and 10mM MgCl
2, iii) 50mM NaCl, 10mM Tris HCl (pH8.5), 1mM DTT and 10mM MgCl
2, iv) 100mM NaCl, 50mM TrisHCl (pH8.3-8.5), 1mM DTT and 10mM MgCl
2Or v) 100mM NaCl, 50mM TrisHCl (pH8.5), 1mM DTT and 6mM MgCl
2Tru9I is containing 100mM NaCl, 50mM TrisHCl (pH8.5-9.25), 10mM MgCl
2With after 30 thermal cyclings, keep medium activity in the Laemmli buffer system Laemmli of 1mM DTT.Similar above-mentioned experiment shows that Tsp509 I is containing 50mM NaCl, 10mM TrisHCl (pH9.0～10), 10mM MgCl
2With keep medium activity through 30 after taking turns thermal cycling in the buffering system of 1mM DTT.
Embodiment 2
Evaluation with restriction endonuclease and archaeal dna polymerase thermotolerance/activity and PCR compatible buffers system
Mensuration can be used to keep the consistency that BstNI patience/active damping fluid scope (above-mentioned) is also evaluated it and used the PCR of primer 5BKIT or 5BKIW and 3KiE.Foundation contains genome K562 DNA (800ng), 30pmol 5 BKIT or 30 pmol 5BKIW, 30pmol 3KiE, and the PCR mixture of each 100 μ M of dNTP (dATP, dCTP, dTTP, dGTP) is used for various buffering systems.The Taq archaeal dna polymerase of 4 units (5 units/μ l; AmpliTaq, perkin elmer) and Taq Start
TM(0.16 μ l is in the antibody dilution buffer of 3.8 μ l for antibody; Clontech) mixing to produce whole mol ratio is 1: 5 Taq archaeal dna polymerase: TaqStart
TMAntibody.Taq archaeal dna polymerase: Taq Start
TMMixtures of antibodies before adding said mixture in room temperature incubation 15 minutes.Total reaction volume is 100 μ l.Reaction places GeneAmp PCR system 9600 (perkin elmer), 94 ℃ of sex change 2 minutes then through 60 ℃ 1 minute, 92 ℃ 20 seconds 30 take turns circulation.Thermal cycling is reflected at 60 ℃ later on and kept 15 minutes.
There is not operation subsequently, by 5%Nusieve GTG gel (FMCBioproducts, Rockland, MD) 28 μ l aliquots containigs of each reaction of electrophoretic analysis on, gel Stratagene Eagle Eye II Video system photograph.With primer 5BKIT and 3KiE, or the efficient of 5BKIW and 3KiE amplification is judged to be low, medium or high.These design of primers combine with restriction endonuclease Bst NI and are used for multiple REMS-PCR system.Bst NI activity/stable on heating analysis is finished in identical reaction buffer with PCR and experienced identical thermal cycling process.Detect two kinds of analytical resultss and not only allow effective pcr amplification but also keep the active condition of restriction endonuclease (table 6) to find out.
Table 6
| Ealkaline buffer | Extra reagent | Pcr amplification efficient | The BstNI activity | ||
| 5BKIT 3KiE | 5BKIW 3KiE | 15 circulations | 30 circulations | ||
| NEB2 | High | High | Medium | Low | |
| NEB3 | High | Medium | Fully | Medium | |
| PCR damping fluid II | 3mM?MgCl 2 | High | High | Medium | Low |
| 6mM?MgCl 2 | High | High | Medium | Low | |
| 10mM?MgCl 2 | High | High | High | Medium | |
| The Stoffel damping fluid | 3mM?MgCl 2 | High | High | Medium | Low |
| 6mM?MgCl 2 | High | High | Medium | Low | |
| 10mM?MgCl 2 | Medium | Medium | Medium | Low | |
| MTris?10?pH8.3 | 10mM?MgCl 2 | High | High | High | Low |
| HTris?50?pH8.3 | Medium | Medium | Fully | Medium | |
| MTris?10?pH8.0 | 10mM?MgCl 2 | High | Medium | Medium | Low |
| MTris?10?pH8.3 | High | High | High | Low | |
| MTris?10?pH8.5 | High | Medium | Medium | Low | |
| HTris?50?pH8.0 | 10mM?MgCl 2 | Medium | Medium | Fully | Medium |
| HTris?50?pH8.3 | Medium | Medium | Fully | Medium | |
| HTris?50?pH8.5 | Low | Medium | Fully | Medium | |
| HTris?50?pH8.3 | 3mM?MgCl 2 | High | Medium | High | Medium |
| 6mM?MgCl 2 | High | Medium | Fully | Medium | |
| 0mM?MgCl 2 | Medium | Medium | Fully | Medium | |
| HTris?50?pH8.3 6mM?MgCl 2 | - | High | Medium | Fully | Medium |
| DTT | High | Medium | High | Medium | |
| aBSA | High | Medium | High | Medium | |
| Non-aBSA | High | Medium | High | Medium | |
| DTT+aBSA | High | Medium | Medium | Medium | |
| The non-aBSA of DTT+ | High | Medium | Medium | Low | |
| Glycerine | Low | Low | High | Medium | |
| T4 gene 32 protein | High | Low | Low | Inactivation | |
Select i simultaneously) cause with primer to the high-level efficiency amplification of 5BKIT and 3KiE and with primer to the amplification of the mid-efficiency of 5BKIW and 3KiE with ii) keep after taking turns thermal cycling completely Bst NI active and keep the buffer conditions of medium activity needing simultaneously to be used for DNA Taq polysaccharase and the active REMS-PCR analysis of Bst NI after taking turns thermal cycling through 30 through at least 15.The buffer conditions that is fit to these standards is 100mM NaCl, 50mM TrisHCl pH8.3 and 6mMMgCl
2
Embodiment 3
Use the REMS-PCR of Bst NI and DNA Taq polysaccharase: in the multiplicated system that adds internal contrast to the analysis of K-ras gene codon 12.
The REMS-PCR method is used to detect the point mutation of K-ras oncogene codon 12.The human cell is Calu I[ATTC HTB54] and K562[ATCC CCL243] available from American type culture collection, Calu I is a kind of lung adenocarcinoma cell, it is heterozygosis at K-ras codon 12, not only had wild-type (GGT) but have mutant (TGT) sequence (D.J.Capon 1983 natures 304,507-513), K562 is an a kind of human leukaemia cell system, its K-ras codon 12 is the (R.L.Ward etc. of wild-type, molecular pathology 1995,48, M273-277).From CaluI and K562, extract genomic dna with standard technique (Sambrook etc. 1989).DNA sample primer 5BKIT, 5BKIW, 3MKiC and 3KiE (table 7) increase by REMS-PCR.
Table 7
| Primer | Function | Sequence |
| ??5BKIT | Diagnostic primers | TATAAACTTGTGGTAGTTGGACCT |
| ??5BKIW | PCR contrasts primer | TTTTGTCGACGAATATGATCC |
| ??3MKiC | BstNI contrasts primer | CTGTATCAAAGCTTGGTCCTGGACCAG |
| ??3KiE | 3 ' primer | CTCATGAAAATGGTCAGAGAAAC |
Boldface type C among the primer 5BKIT is mispairing with respect to the K-ras gene order, if codon 12 is a wild-type, this mismatched bases causes importing BstM identification/cleavage site in the K-ras amplicon.The amplicon that contains at the 1st or the 2nd coding mutation of codon 12 does not contain Bst M identification/cutting sequence.Primer 5BKIT and 5BKIW also produce by pcr amplification of similar mark with biotin labeling at its 5 ' end.The boldface type G of primer 3MKiC is mispairing with respect to the K-ras sequence, and this causes importing BstM identification/cleavage site and will being incorporated in any amplicon of the generation with this primer and 5BKIT or 5BKIW amplification in that primer is inner.
1: 10 mixture (weight ratio) of the genomic dna of K562, Calu I and Calu I:K562 is with the amplification of multiple REMS-PCR system, and reaction contains in 100mMNaCl, 50mM Tris (pH8.3) and 6mM MgCl
2In genomic dna (800ng), 30pmol 5BKIT, 30pmol 3KiE, 5pmol 5BKIW, 80pmol3MKiC, each 100 μ M of dNTP (dATP, dCTP, dTTP, dGTP), the Bst NI of 80 units (10 units/μ l, New England Biolabs) and 4 Taq of unit archaeal dna polymerases (5 units/μ l, Ampli Taq, perkin elmer).Total reaction volume is 100 μ l, two control reaction or contain CaluI DNA or contain dH
2O (no DNA) and all do not have Bst NI and exist.Reaction places Gene Amp PCR system 9600 (perkin elmers), 94 ℃ of sex change carry out after 3 minutes 60 ℃ 1 minute succeeded by 92 ℃ 20 seconds 30 take turns circulation, the thermal cycling afterreaction was kept 15 minutes in 60 ℃.
25 μ l products of each reaction do not have operation subsequently and (FMC Bioproducts, Rockland MD) go up electrophoresis to be analyzed, and gel is taken a picture with Polaroid (Polaroid Land) camera in 5% Nusieve GTG gel.Containing CaluI DNA but do not have in the control reaction of BstNI, three fragments are high-visible; The fragment that comprises the 185bp that mixes primer 5BKIT and 3KiE amplicon comprises the 156bp fragment of the amplicon that mixes primer 5BKIT and 3MKiC and comprises the 114bp fragment of the amplicon that mixes primer 5BKIW and 3KiE.As seen a bar segment that comprises the 85bp that mixes primer 5BKIW and 3MKiC amplicon faint.
In the reaction that contains Bst NI, the segmental existence of 185bp is used to diagnose the existence of K-ras codon 12 sudden changes, this fragment is found in the reaction of the CaluI:K562DNA that contains Calu I and 1: 10 ratio, but in the reaction that only contains K562DNA, can not see, the Bst NI contrast fragment of 156bp (and 85bp) all can not be seen in any reaction that contains Bst NI, and this proof Bst NI can mediate the inhibition fully to second fragment amplification.Because the amplicon of any 156bp will contain Bst NI site, the inhibition of this fragment amplification does not rely on the sudden change situation of codon 12.Restriction endonuclease contrasts segmental disappearance and can do clearly to judge to negative findings.During the PCR contrast fragment of 114bp was found in and comprises that the institute that contains K562 DNA reaction responds, this confirmed reaction conditions, comprises that the template DNA amount is enough to increase by PCR.PCR contrasts segmental existence and can do clearly to judge to positive findings.In the reaction that does not contain DNA, do not see any fragment.
Embodiment 4
REMS-PCR: the limit that point mutation detects
Contain with the codon 12 of K-ras gene in the sample of the Calu I DNA of Sup TI DNA dilution by analysis with the limit of REMS-PCR check point sudden change and to be evaluated.Sup TI[ATCC CRL 1942] be a kind of leukemia cell system available from American type culture collection.Calu I is heterozygous mutant of K-ras codon 12 and Sup TI is the wild-type of K-ras gene codon 12.From these clones, extract genomic dna and increase with standard technique (Sambrook etc. 1989) with REMS-PCR.CaluI DNA with Sup TIDNA be diluted to 1: 10,1: 10
2, 1: 10
3, 1: 10
4, 1: 10
5With 1: 10
6Calu I: the ratio of Sup T1 (weight ratio).
The REMS-PCR reaction contains at 100mM NaCl, 50mM Tris (pH8.3) and 6mMMgCl
2In genomic dna (1 μ g), 30pmole 5BKIT, 30pmole 3KiE, 5pmole 5 BKIW, the Bst NI of dNTP (dATP, dCTP, dTTP, dGTP) each 100mM and 40 units (10 units/μ l, New England Biolabs).Taq archaeal dna polymerase (5 units/μ l with 4 units; AmpliTaq, perkin elmer) and TaqStart
TM(0.16 μ l is in the antibody dilution buffer of 3.8 μ l; Clontech) mixing to produce whole molar ratio is 1: 5 Taq archaeal dna polymerase: TaqStart
TMTaqDNA polysaccharase: Taq Start
TMMixtures of antibodies before adding the PCR mixture in room temperature incubation 15 minutes.Total reaction volume is 100 μ l.Reaction places Gene Amp PCR system 9600 (perkin elmers), 94 ℃ of sex change carry out after 2 minutes 60 ℃ 1 minute succeeded by 92 ℃ 20 seconds 30 take turns circulation.Thermal cycling is reflected at 60 ℃ later on and kept 15 minutes.
28 μ l sample aliquot of each reaction are without subsequently operation and by 5%NusieveGTG gel (FMC Bioproducts, Rockland, MD) electrophoresis on is analyzed, and gel is taken a picture with Polaroid land (Polaroid) camera and Stratagene Eagle Vision II imaging system.The 185bp fragment that produces with primer 5BKIT and 3KiE amplification is used for existing of diagnostic code 12 sudden changes, this fragment is taken a picture by Polaroid and the hawkeye imaging be found in contain 1: 10,1: 10
2With 1: 10
3The Calu I of ratio: be found in the reaction of Sup T1 DNA and by the hawkeye imaging and contain 1: 10
4In the reaction of ratio.This 185bp fragment neither sees and contains 1: 10
5With 1: 10
6The Calu I of ratio: also do not see in the reaction that only contains Sup T1 in the reaction of Sup T1DNA.The PCR contrast fragment of the 114bp that produces with primer 5BKIW and 3KiE fragment amplification be found in responded, this proved response condition comprises that the amount of template DNA is enough to increase effectively by PCR.
Colorimetric analysis has also been carried out in REMS-PCR reaction, this analysis classes be similar to the colorimetric analysis that Findlay etc. describes (clinical chemistry, 1,993 39/9,1927-1933).Pcr amplification is by hybridizing on the oligonucleotide probe and caught by specificity, and this probe is covalently attached on the latex bead that is applied to Periodontal Surecell blank space separation point position.The sequence of capture oligo and the specific PCR amplicon that captures are listed as follows (table 8).K-Capl and K-Cap2 are designed to only catch the diagnostic K-ras amplicon that produces through the sudden change template amplification with primer 5BKIT and 3KiE specifically.K-Cap3 is designed to catch the amplicon that produces through sudden change or wild-type template amplification with 5BKIT or 5BKIW and 3KiE.H-Capl catches non-specific amplification and the negative control of non-specific amplification or hybridization is provided.
Table 8
| Probe (function) | Sequence | Homology clip size (primer that mixes) | The amplicon type of catching |
| K-Capl (diagnosis) | TAGCTGTATCGTCAAG GCACTCTT | 185bp(5BKIT/3KiE) | Muton only |
| K-Cap2 (diagnosis) | AAATGATTCTGAATTA GCTGTATCGTC | 185bp(5BKIT/3KiE) | Muton only |
| K-Cap3 (PCR contrast) | GCACCAGTAATATGCA TATTAAAACAAG | 185bp(5BKIT/3KiE) 114bp(5BKIW/3KiE) | The muton wild-type |
| H-Capl (negative control) | ACCATCCAGCTGATCC AGAACCAT | Nil | Non-specific |
The aliquots containig of 4 kinds of oligonucleotide latex bead (being scattered in 1.6 μ l10mM Tris with 0.25%, among the 1mMEDTA pH7.4) is applied to the different loci on the Surecell film, and all 4 kinds of oligonucleotide are arranged in each Surecell hole.Allow dry 15 minutes of oligonucleotide latex bead.The aliquots containig of 30 each PCR of μ l is with KCl, 10mM Tris (pH8.3) and the 10mMMgCl of 170 μ l 50mM
2Dilution.Solution was in 95 ℃ of sex change 6 minutes and put on the Surecell hole.Thereby Surecells made pcr amplification and capture oligo hybridization in 5 minutes in 50 ℃ of incubations then.With 300 μ l50mM KCl, 10mM Tris (pH8.3) and 10mM MgCl
2In 50 ℃ of hole flushings.The streptomycete avidin conjugate that the amplicon of hybridization is attached to horseradish peroxidase (EC1.11.1.7) with three reacts and in room temperature incubation 2 minutes.It is minimum that the repeated washing step is reduced to nonspecific reaction.Add 4 Leucodye/H
2O
2And Surecell was in room temperature incubation 2 minutes.The fixed mixture is used as catalyzer the oxidisability of dye molecule from colourless to blue form transforms.Reaction is with 4 0.1%NaN
3Stop.The coloured speckle that obtains is by scoring with naked eyes with the color table comparison and being classified as 0 (colourless) to 10 (dark blue) (tables 9)
Table 9
| The color score | |||||||
| CaluI：SupTIDNA | 1∶10 | 1∶10 2 | 1∶10 3 | 1∶10 4 | 1∶10 5 | 1∶10 6 | SupTl |
| K-Cap 1 (muton is specific) | 9 | 8 | 4 | 2 | 0 | 0 | 0 |
| K-Cap 2 (muton is specific) | 9 | 8 | 4 | 2 | 0 | 0 | 0 |
| K-Cap 3 (PCR contrast) | 9 | 9 | 9 | 9 | 9 | 9 | 9 |
| H-Capl (non-specific negative control) | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
When by gel electrophoresis or colorimetric analysis, the susceptibility of REMS-PCR method is when being present in 1: 10
3～: 1: 10
4Allow detection in the time of in the background of wild-type sequence to the K-ras codon 12 muton sequences of selective amplification.Wild-type K-ras codon 12 sequences do not detect in this REMS-PCR analyzes.Document shows that the susceptibility of this level will be enough to analyze the DNA that extracts from clinical samples, comprises cutting tissue and vivisection, cytological sample and body fluid/secretory product such as ight soil, urine and contain the phlegm of a small amount of exfoliation tumour cell.
Clinically, a large amount of samples are analyzed simultaneously, and it is necessary that amplification does not begin in advance, because this can cause non-specific product to comprise the amplification of primer dimer.Monoclonal anti cognition is attached on the DNA Taq polysaccharase, thereby suppresses active and amplification before first denaturing step.Initial with in the experiment of REMS-PCR, 1: 28 the DNA Taq polysaccharase of recommending by Clontech: Taq Start
TMThe standard mol ratio cause false positive results owing to the amplification of wild-type Sup Tl dna profiling, detected various mol ratios and established DNA Taq polysaccharase: Taq Start
TMThe low mol ratio of antibody is as the formation that can suppress non-specific amplification and primer dimer at 1: 5 and the non-false positive result.
Embodiment 5 usefulness REMS-PCR are to the analysis of clinical samples
Method (Sambrook etc. 1989) with standard is extracted genomic dna from normal mucous membrane of colon (NC) and adenocarcinoma of colon (CA).Existence with K-ras codon 12 sudden changes in the REMS-PCR analytic sample that has the following step variation among the embodiment 4; DNA (0.5 μ g) increases in the presence of 4 Taq of unit archaeal dna polymerases and the 80 Bst NI of unit.Pass through 5%Nusieve GTG gel (FMC Bioproducts, Rockland, MD) 30 μ l sample aliquot of each reaction of electrophoretic analysis on without operation subsequently.
Sudden change exists in 185bp fragment diagnostic code 12 that produces with primer 5BKIT and 3KiE amplification, by gel electrophoresis visible this fragment in two reactions of the DNA that contains gland cancer sample CA7 and CAg, this diagnostic fragment fails to see in other two reactions of the DNA that contains gland cancer sample CA1 and CA2, also fails to see in 4 reactions that contain the DNA that extracts from normal mucous membrane of colon NC1, NC2, NC7 and NC8.The contrast fragment of the 114bp that produces with primer 5BKIW and 3KiE amplification is found in all reactions, shows that effective pcr amplification betides in all reactions.
Colon's genomic dna passed through enrichment PCR (R.L Ward etc., molecular pathology 1,995 48, M273-277) existence of analysis K-ras codon 12 sudden changes of standard in the past.The amplification of carrying out with REMS-PCR or enrichment PCR has then obtained consistent result with gel electrophoresis analysis.Two kinds of PCR method show that the DNA of gland cancer sample CA7 and CA8 has the sudden change of K-ras codon 12 and the DNA of gland cancer CA1 and CA2 and normal mucous membrane sample NC1, NC2, NC3 and NC4 is wild-types in codon 12 positions.These results prove that REMS-PCR is suitable for the real-time analysis of clinical samples.Embodiment 6:REMS-PCR: a kind of system that allows to identify the replacement of specificity Nucleotide
The REMS-PCR system is used to detect the point mutation of K-ras oncogene codon 12, and the extra analysis with restriction endonuclease has not only confirmed the diagnosis of codon 12 sudden changes but also allowed to identify that special Nucleotide replaces.The human cell is CaluI[ATCC HTB54], A549[ATCC], K562[ATCC CCL243] and, Sup T1[ATCC CRL 1942] available from American type culture collection.Calu I is a kind of lung adenocarcinoma cell, and it is heterozygosis on K-ras codon 12, not only had wild-type (GGT) but also have sudden change (TGT) sequence (D.J.Capon1983 nature 304,507-513).A549 be a kind of K-ras codon 12 homozygous mutation (AGT) lung adenocarcinoma cell (D.M.Valenzuela and J.Groffen1986 NAR 14,843-852).K562 and Sup T1 are the leukemia cell system of K-ras codon 12 for wild-type.From these clones, extract genomic dna with standard technique (Sambrook etc. 1989).
REMS-PCR finishes with primer 5BKIT and 3AKIP, and it imports a plurality of restriction endonuclease identification/cleavage sites simultaneously.Primer 5BK5 and 3K6 work as PCR contrast primer.(table 10)
Table 10
| Primer | Sequence: import that Restriction Enzyme is the site represents (extra base mismatch is represented with underscore) with the base K-ras gene mismatch with black matrix |
| 5BKIT | TATAAACTTGTGGTAGTTGGACCT |
| 3AKIP | G GA TGAC TCA TTAAGGCACTCTTGCCTACGCCC |
| 5BK5 | TCAGCAAAGACAAGACAGGTA |
| 3K6 | AGCAATGCCCTCTCAAGA |
Primer 5BKIT causes at codon 12 for importing Bst NI identification/cleavage site in the K-ras amplicon of wild-type.Primer 3AKIP induces identification/cleavage site of one or more restriction restriction endonuclease BsaJI, StyI, AvrII, MnlI, AciI, RleI and Bsu36I, (table 11) as follows in the K-ras amplicon of codon 12 sudden changes.Table 11
| K-ras sequence and inductive restriction enzyme identification/cleavage site (are represented with black matrix by the base mismatch that 5BKIT (C) and 3AKIP (G) import, the point mutation of codon 12 is represented with underscore, N=T or A or C or G) | ||||
| Codon 11 | Codon 12 | Codon 13 | Restriction enzyme | |
| Wild-type sequence | CCT CCT | GGG GG | GGC | Bst?NI |
| The muton sequence | CCN | NGG | Bsa?JI | |
| CCT | TGG | Sty?I | ||
| CCT | AGG | AvrII/Sty?I | ||
| CCT | C | Mnl?I | ||
| G CG | G | Aci?I | ||
| T | G TG | GG | Rle?AI | |
| CCT | N AG G AG | G G | Bsu?36I Mnl?I | |
The sudden change amplicon is listed in table 12 to the actual sudden change that the expectancy model of restriction endonuclease Bsa JI, StyI, AvrII, MnlI, AciI, RleI and Bsu 36I cutting susceptibility and resistance depends on codon 12.Table 12
| Codon 12 positions 1 and 2 (point mutation is represented with underscore, N=T or A or C) | The restriction endonuclease of cutting sudden change amplicon | Do not cut the restriction endonuclease of sudden change amplicon |
| ????NG | Bsa?JI | |
| ????TG | Bsa?JI/Sty?I | Avr?II |
| ????AG | Bsa?JI/Sty?I/Avr?II | |
| ????CG | Bsa?JI/Mnl?I | Bsu?36I |
| ????GN | Bsa?JI | |
| ????GT | Rle?AI | Bsa?JI |
| ????GC | Aci?I | Bsa?JI |
| ????GA | Mnl?I/Bsu?36I | Bsa?JI |
The human cell is that the genomic dna of CaluI, A549, K562 and SupTl increases in multiple REMS-PCR system, reacts on 100mM NaCl, 50mM Tris (pH8.3) and 6mM MgCl
2In contain the Bst NI (10 units/μ l, New England Biolabs) of genomic dna (500ng), 50pmole 5BKIT, 50pmole3AKIP, 3pmole 5BK5, each 100mM of 3pmole 3K6, dNTP (dATP, dCTP, dTTP, dGTP), 40 units.The Taq archaeal dna polymerase of 4 units (5 units/μ l; Ampli Taq, perkin elmer) and Taq Stat
TM(0.06 μ l is in 1.5 μ l antibody dilution buffers, and Clontech) mixing to produce whole mol ratio is 1: 2 Taq archaeal dna polymerase: Taq Start for antibody
TMAntibody.Before adding reaction with the Taq archaeal dna polymerase: Taq Start
TMMixtures of antibodies was in room temperature incubation 15 minutes.Total reaction volume is 100 μ l.Reaction places Gene Amp PCR system 9600 (perkin elmers), 94 ℃ of sex change 3 minutes and carry out then 60 ℃ 1 minute then 92 ℃ 20 seconds 30 take turns circulation.Thermal cycling reacts on 60 ℃ later on and kept 15 minutes.
20 μ l aliquots containigs of each reaction without subsequently operation by (MD) electrophoresis on is analyzed for FMC Bioproducts, Rockland, and gel is taken a picture with Polaroid (polaroid land) camera in the 5%NusieveGTG gel.Existing of fragment diagnostic code 12 sudden changes of the 58bp that produces with primer 5BKIT and 3AKIP amplification, this fragment is found in the reaction that contains Calu I and A549 DNA, but fails to see in the reaction that contains SupTl or K562 DNA.The PCR contrast fragment of the 167bp that produces with primer 5BK5 and 3K6 amplification is present in all reactions, comprises in the reaction that contains SupTl and K562DNA.This confirmed effective pcr amplification appears in responding.
The 15 μ l aliquots containigs that contain CaluI or A549DNA reaction are with 10 unit limit endonuclease BsaJI, StyI, AvrII, MnlI or AciI (as shown in table 13 below) digestion and the suitableeest digestion temperature incubation that illustrates in manufacturers (New England Biolabs).(MD) electrophoresis on is analyzed and gel Polaroid camera photograph for FMC Bioproducts, Rockland with 5%Nusieve GTG gel in reaction.Table 13
| Template DNA | Produce the primer of amplicon | Restriction endonuclease | The result | Codon 12 positions 1 and 2 sequence (N=A, C or T) |
| K562 | 5K5/3K6 only | - | - | Wild-type-GG |
| SupTl | 5K5/3K6 only | - | - | Wild-type-GG |
| CaluI | 5BKIT/3AKIP 5K5/3K6 | Bsa?JI Sty?I Avr?II | Cutting cutting resistance | The non-AG result of muton NG TG or AG: muton (TG) |
| A549 | 5BKIT/3AKIP 5K5/3K6 | Bsa?JI Sty?I Avr?II | Cutting cutting cutting | Muton NG AG or TG AG result: muton (AG) |
This REMS-PCR system allows the detection to 12 sudden changes of K-ras oncogene codon.The evaluation that restriction endonuclease analysis has subsequently confirmed the existence of sudden change and allowed specific nucleotide is replaced.
Embodiment 7: the REMS-PCR system that uses Bst NI and Stoffel polysaccharase
The human cell is Calu I[ATCC HTB54] and SupT1[ATCC CRL 1942] genomic dna increase with REMS-PCR.Extract genomic dna with standard techniques (Sambrook etc. 1989) from these clones, DNA increases with REMS-PCR, contains in the reaction to be dissolved in 10mMKCl, 10mM Tris (pH8.3) and 10mM MgCl
2(1 * Stoffel damping fluid; Perkin elmer) 5BKIW of the genomic dna in (1 μ g), 30pmol 5BKIT, 30pmole 3KiE, 2pmole, each 100mM of dNTP (dATP, dCTP, dTTP, dGTP), with the Bst NI (10 units/μ l, New England Biolabs) of 40 units, control reaction does not contain DNA (dH
2O).Stoffel fragment (10 units/μ l with 5 units; Perkin elmer) with Taq antibody TP4 (1994 biologies such as D.J.Sharkey/technology 12,506-509) (0.05 μ l is in the Clontech antibody dilution buffer of 1.2 μ l) to mix to produce whole mol ratio be 1: 2 Stoffel fragment: Taq antibody TP4.Before adding reaction with the Stoffel fragment: the Taq mixtures of antibodies was in room temperature incubation 15 minutes.Total reaction volume is 100ul.Reaction places Gene Amp PCR system 9600 (perkin elmers), 94 ℃ of sex change 2 minutes and carry out then 60 ℃ 1 minute then 92 ℃ 20 seconds 30 take turns circulation.Thermal cycling reacts on 60 ℃ later on and kept 15 minutes.
The sample aliquot of every kind of reaction of 25 μ l is passed through the 5%NusieveGTG gel without operation subsequently, and (MD) electrophoresis on is analyzed for FMC Bioproducts, Rockland, and gel is taken pictures with the Polaroid camera.Existing of 185bp fragment diagnostic code 12 sudden changes that produce with primer 5BKIT and 3KiE amplification, this fragment is found in the reaction that contains Calu I DNA, but do not see in the reaction that contains Sup TI DNA, the 114bp PCR contrast fragment that produces with primer 5BKIW and 3KiE amplification is found in both reactions, shows that PCR increases effectively.Do not have fragment to be found in and do not contain in the control reaction of template.
Embodiment 8: the REMS-PCR system that uses Bsl I and Taq archaeal dna polymerase
The REMS-PCR analytical procedure is developed to detect the point mutation of K-ras oncogene codon 12.In this analysis, if amplicon is a wild-type at codon 12, they contain identification/cutting sequence of heat resistant restriction endonuclease Bsl I.The amplicon that contains sudden change at first or second Nucleotide of codon 12 does not contain identification/cutting sequence of Bsl I.
The human cell is CaluI[ATCC HTB54] and K562[ATCC CCL243] genomic dna increase with REMS-PCR.Calu I is wild-type for heterozygous mutant K562 at codon 12 at the codon 12 of K-ras gene.From these clones, extract genomic dna with standard technique (Sambrook etc. 1989), Calu I DNA with K562 DNA be diluted to 1: 10,1: 10
2, 1: 10
3Calu I:K562 ratio (weight).
DNA increases by REMS-PCR with primer 5BKIQ, 5BKIW and 3KiH (table 14).2 runic C among the 5BKIQ are mispairing with respect to the sequence of K-ras gene, and these mismatched bases cause at codon 12 for introducing Bsl I site in the amplicon of wild-type.Primer 5BKIQ and 5BKIW are biotin labeled.
Table 14
| Primer | Sequence |
| ????5BKIQ | TATAAACTTGTGGTACCTGGAGC |
| ????5BKIW | TTTTGTCGACGAATATGATCC |
| ????3KiH | GAAAATGGTCAGAGAAACC |
React to contain and be dissolved in 100mM NaCl, 50mM Tris (pH8.5), 1mM DDT and 6mM MgCl
2In each 100 μ M of genomic dna (400ng), 30pmole 5BKIQ, 15pmole3KiH, 0.5pmole 5BKIW, dNTP (dATP, dCTP, dCTP, dTTP, dGTP) and the Bsl I (50 units/μ l, New England Biolabs) of 10 units.Taq archaeal dna polymerase (5 units/μ l with 8 units; Ampli Taq, perkin elmer) and Taq Start
TM(0.16 μ l is in the antibody dilution buffer of 3.8 μ l for antibody; Clontech) mixing to produce whole mol ratio is 1: 5 Taq archaeal dna polymerase: Taq Start
TMAntibody.Taq archaeal dna polymerase: Taq Start
TMMixtures of antibodies before adding reaction in room temperature incubation 15 minutes.Total reaction volume is 50 μ l.Reaction places Gene Amp PCR system 9600 (perkin elmers) and in 94 ℃ of sex change 2 minutes.Reaction is carried out 10 then and is taken turns 63 ℃ of then 92 ℃ of circulations of 20 seconds and 20 take turns 55 ℃ of then 92 ℃ of circulations of 20 seconds in 1 minute then in 30 seconds.Thermal cycling reacts on 55 ℃ later on and kept 15 minutes.
28 μ l sample aliquot of every kind of reaction are without subsequently operation, and (MD) electrophoresis on is analyzed for FMC Bioproducts, Rock/and, and gel is taken a picture with the Polaroid camera by the 5%NusieveGTG gel.Existing of fragment diagnostic code 12 sudden changes of the 180bp that produces with primer 5BKIQ and 3KiH amplification, this fragment is found in and contains 1: 10 and 1: 10
2The Calu I of ratio: in the reaction of K562.The diagnostic fragment of this 180bp is failed to see and is contained 1: 10
3Ratio Calu I: in the reaction of Sup T1 or only contain in the reaction of K562.The PCR contrast fragment of the 109bp that produces with primer 5BKIW and 3KiH amplification is found in and shows through PCR in all reactions and increase effectively.
This system detects the sudden change of K-ras codon 12 with restriction endonuclease Bsl I, this restriction endonuclease can be used for detecting the systems that betide any one codon 12 among 3 ras-oncogene K-ras, H-ras and the N-ras or 13 sudden changes, and it also can be used for analyzing other sudden change that betides coding glycine or proline(Pro) codon or other sudden change that betides Nucleotide C or G.
Embodiment 9: the REMS-PCR method by needs BstNI digestion subsequently is to the analysis of K-ras codon 12
A kind of alternative method can be used for detecting the point mutation of K-ras oncogene codon 12.With standard techniques (Sambrook etc. 1989) from Calu I[ATCC HTB54] and K562[ATCC CCL243] extract genomic dna.Calu I DNA uses K562 DNA with 1: 10,1: 10
2, 1: 10
3With 1: 10
4Calu I: K562 dilutes than (weight).DNA sample apparatus has the primer 5BKIM of sequence GACTGAATATAAACTTGTGGTAGTTGGACCT and has sequence
GG
ATG
ACTCAT
TThe primer 3AKIL amplification of TTCGTCCACAAAATGATTCTGAATTAG.If the runic C in primer 5BKIM is mispairing and they at codon 12 for wild-type then cause introducing identification/cleavage site of Bst NI in the K-ras amplicon with respect to the K-ras gene order.Rule below with the K-ras mismatched bases among the 3AKIL.
10 * PCR damping fluid the II (perkin elmer), the 1.5mMMgCl that in total reaction volume is the reaction of 100 μ l, contain each 100 μ M, 10 μ l of genomic dna (800ng), 40pmole 5BKIM and 40pmol 3AKIL, dNTP (dATP, dCTP, dTTP, dGTP)
2, 80 units Bst NI (10 units/μ l, New England Biolabs) and 2 Taq of unit archaeal dna polymerase (5 units/μ l; Ampli Taq, perkin elmer).Reaction places Gene Amp PCR system 9600 (perkin elmers), 94 ℃ of sex change 3 minutes and carry out 40 then and take turns 60 ℃ of then 92 ℃ of circulations of 20 seconds in 1 minute.Thermal cycling reacts on 60 ℃ later on and kept 15 minutes.
The sample aliquot of every kind of reaction solution of 25 μ l is analyzed without operation subsequently.Second 25 μ l sample aliquot of every kind of reaction in the total reaction volume of 35 μ l with the 15 Bst NI of unit (10 units/μ l, New England Bioolabs), 10X NEB2 damping fluid (New England Biolabs) incubation of 100 μ g/ml bovine serum albumins (New England Biolabs) and 3.5 μ l.These reactions cover and are incubated overnight with the mineral oil of 20 μ l and in 60 ℃.(MD) analyzed and taken a picture with the Polaroid camera for FMC Bioproducts, Rockland by the electrophoresis on by 5%Nusieve GTG gel for respond.
In being responded with the institute of Bst NI digestion after the PCR, as seen with primer 5BKIM and 3AKIL amplification the fragment of the 103bp of generation only be found in contain 1: 10,1: 10
2With 1: 10
3Ratio Calu I: in the reaction of K562 DNA.The 103bp fragment is failed to see and is contained 1: 10
4The Calu I of ratio: also fail in the reaction of K562 DNA to see in the reaction that only contains K562DNA.The 73bp fragment that is produced by Bst NI digestion wild-type amplification is found in all reactions.In the reaction with Bst NI digestion after PCR, the segmental existence of 103bp can be diagnosed the existence of K-ras codon 12 sudden changes.
When with 1: 10
3The Calu I of ratio: the susceptibility of this kind method allowed the detection to sudden change Calu I DNA when K562 DNA existed.Under these reaction conditionss, comprise that in PCR reaction Bst NI causes preferential amplification (enrichment) mutant nucleotide sequence but do not cause the inhibition fully of wild-type K562 sequence amplification.Therefore be reflected at the digestion of using Bst NI before preferably analyzing.This method have the REMS-PCR method of standard enrichment PCR method (need two take turns PCR add that the digestion of intermediary restriction endonuclease is with the enrichment mutant nucleotide sequence) and standard (wherein the amplification of wild-type sequence suppressed fully and analyzing before need not subsequently operation as digesting) between the moderate simplicity.
Discuss
The essential i of restriction endonuclease and archaeal dna polymerase in the REMS-PCR method) same reaction conditions that must be compatible with PCR (as, salt, pH) in work and ii) enough heat-resisting in the thermal cycling process of PCR needs, to keep activity in these reaction conditionss.Can identify that following will being applicable to mix in the REMS-PCR method if list in some restriction endonuclease of table 1 and other heat resistant restriction endonuclease buffer conditions, described buffer conditions is i) with the restriction endonuclease activity compatible and when react, keep endonuclease activity and ii) compatible and when keeping polymerase activity in the PCR process during thermal cycling with the dna polymerase activity of while for the thermal cycling in the PCR process.
Knew little about it owing in the past restriction endonuclease is kept active ability in the required thermal cycling process of PCR, so develop a kind of simple and easy to do analytical procedure to identify candidate's heat resistant restriction endonuclease and reaction conditions.In activity/thermotolerance was analyzed, the enzymic activity of restriction endonuclease can be compared later in the thermal cycling of certain number in various reaction conditionss.In this analytical procedure, preparation contains the reaction solution of primer, dNTP and the archaeal dna polymerase of Standard PC R concentration.Do not contain template DNA in the reaction solution but comprise Laemmli buffer system Laemmli, have or do not have additional agents and restriction endonuclease to be measured.Reaction places thermo cycler, rises to high temperature thermal cycling then, and termination reaction through the thermal cycling of certain number after is in the plasmid DNA adding pipe and be reflected at incubation under the optimum temperuture of the specified restriction endonuclease of manufacturers.The enzymic activity of restriction endonuclease can be evaluated by the cutting degree of observing plasmid DNA in the gel electrophoresis.
Activity/thermotolerance Analysis and Identification goes out various restriction endonuclease, comprises Bst NI, BslI, Tru91 and Tsp 509I, they in some buffer conditions, be enough to heat-resisting with keep appropriateness later in the essential thermal cycling of PCR or catalytic activity completely.In the thermal cycling process, keep the most effective reaction conditions of catalytic activity to be identified out.The catalytic activity of restriction endonuclease after thermal cycling is according to the pH of damping fluid and ionic strength, univalent cation (K
+Or N
+) selection and concentration, free Mg
2+Concentration and other additive comprise the existence of dithiothreitol (DTT) (DTT) and change.The influence of every kind of composition depends on other composition in the damping fluid in these compositions.
Also the enzymic activity of very possible restriction endonuclease is kept by the temperature in the reduction PCR process and the time of DNA sex change.The factor of known effect duplex DNA molecule melting temperature(Tm) comprises the existence of salt concn and reagent such as methane amide, methyl-sulphoxide, glycerine and ethylene glycol, and at least some PCR systems of these reagent are compatible.Comprise into these or other influence the DNA melting temperature(Tm) reagent can allow PCR to finish in the denaturation temperature and/or the time of reducing.These reagent also can have direct front or negative impact to the activity and/or the thermotolerance of restriction endonuclease (and/or archaeal dna polymerase).Various thermal cycling temperature curves can be evaluated by above-mentioned thermotolerance/activation analysis method the active influence of restriction endonuclease in the presence of additional agents.Keep activity/thermotolerance detection of the available routine of evaluation of the active reaction conditions of restriction endonuclease to need not creative work and just can finish in extra heat resistant restriction endonuclease and the thermal cycling process.
For REMS-PCR, reaction conditions must not only keep the catalytic activity of restriction endonuclease but also them must be applicable to PCR, so buffer conditions must be compatible with the activity and the thermotolerance of the archaeal dna polymerase of thermal cycling process.Many commercial archaeal dna polymerases that can be used for PCR that provide are arranged, and the general aspects of these enzymes alters a great deal, and comprises their the suitableeest buffer conditions and their condition and ranges that can tolerate.Under the active reaction conditions of known maintenance restriction endonuclease to PCR in the detection of various archaeal dna polymerase efficient allow evaluation to compatible archaeal dna polymerase/restriction endonuclease/damping fluid combination.Proved and kept the active Reaction conditions range of restriction endonuclease has also been analyzed they and PCR with various primers and various archaeal dna polymerase consistency.The heterogeneity of reaction conditions to the influence of PCR according to different primers to and change and responsible other reacted constituent.For this reason, the concrete primer cover of PCR needs should detect by this way.With restriction endonuclease and archaeal dna polymerase activity compatible and cause to be identified through the test that need not creative work of routine effective amplification PCR condition simultaneously with concrete primer.
REMS-PCR needs heat resistant restriction endonuclease identification/cleavage site to cross over the Nucleotide of heritable variation to be analyzed, this site or can produce naturally or can be induced by containing the inner primer with the template mispairing.When restriction endonuclease identification/cleavage site was induced by primer, this site part was arranged in primer and partly is positioned at the composition sequence of amplicon primer 3 '.Therefore, primer must comprise any base mismatch that need be used to induce restriction nuclease enzyme site, but should be not overlapping with base to be analyzed.Design set up in the rule that nearly 3 ' end contains the PCR primer of base mismatch (S.Kwok waits 1990, nucleic acids research 18,999-10005).Reach 100 times though the primer of some terminal mispairing can not increase and reduce the output of specific amplification effectively, great majority will the same amplification effectively with complete paired primer.For example when terminal 3 ' base was G in the primer, it extended on the template that contains C, T or G on the complimentary positions, but does not extend on the template that contains A on the complimentary positions.
When restriction endonuclease only needs the tetranucleotide recognition sequence (as Tru9I or Tsp509I) of a weak point or when they discerned a plurality of sequences (as Bst NI), identification/cleavage site can more easily be induced.Identification/the cleavage site of the restriction endonuclease of the short sequence that identification is interrupted is easy to induce especially, for example, Bsl I recognition sequence CCNNNNNNNGG, wherein N is any Nucleotide.Bsl I can be used for analyzing the sudden change that betides coding glycine (GGN) or proline(Pro) (CCN) codon.Usually, the primer of inducing the BslI recognition site in these codons of design can be extended because they will not need nearly 3 ' terminal base mismatch and be positioned at the single of primer sequence middle part or two bases that do not match can tolerate and also often do not suppress pcr amplification by archaeal dna polymerase.
In addition, those skilled in the art can design the primer that can induce the BslI recognition site and is used to analyze great majority (about 80%) sudden change that betides G or C.The sudden change of bases G and C is very common, for example, the per-cent that betides the p53 sudden change of G or C base is at least 77% in the colorectal carcinoma sudden change, in the lung cancer sudden change, be at least 72%, in the bladder cancer sudden change, be at least 74%, in mammary cancer sudden change, be at least 61% and in the cerebral tumor sudden change, be at least 66% (1996 nucleic acids research 24 such as M.Hollstein, 141-146).Following table is listed around all possible combination of base C or G sequence and is used for inducing in these positions CC or the GG required terminal bases of primer as the part in Bsl I site.Estimate that the template/combination of primers compatible with PCR is shown in table 15.
Table 15
| Template sequence N=A adjacent to target base (underscore), C, G, T X=A, C, T Y=A, G, T | The primer type has justice (5 ' primer) antisense (3 ' primer) | Primer 3 ' base | Template 3 ' base | With the PCR consistency |
| GGN | Justice is arranged | ????G | ????C | Be |
| AGN | Justice is arranged | ????G | ????T | Be |
| CGN | Justice is arranged | ????G | ????G | Be |
| TGX | Justice is arranged | ????G | ????A | Not |
| NGG | Justice is arranged | N=the same with each template (justice is arranged) | N=the same with each template (antisense) | Be |
| NCC | Antisense | ????G | ????C | Be |
| NCT | Antisense | ????G | ????T | Be |
| NCG | Antisense | ????G | ????G | Be |
| YCA | Antisense | ????G | ????A | Not |
| CCN | Antisense | N=the same with each template (antisense) | N=the same with each template (justice is arranged) | Be |
The example of the identification/cleavage site of natural or inductive heat resistant restriction endonuclease is listed in table 16 in the gene relevant with acquired disease, and in these examples, the restriction endonuclease of identification wild-type sequence is identified.This table comprises the known restriction endonuclease compatible with REMS-PCR and other endonuclease possible compatible with present method.The primer that is used for these mutation analysises must comprise needs inductive base (representing with runic) but can not be overlapping with base (underscore) to be analyzed.Ras-proto-oncogene (K-ras, H-ras and N-ras) is frequently activated by the point mutation that obtains codon 12,13 and 61 in a lot of human cancers.So glycine Bsl I can be used to analyze most of ras-sudden changes because the codon 12 and 13 of all three kinds of ras genes is all encoded.A new point mutation among the intron D of H-ras also is found in the bladder cancer.The HIV strain is relevant with the acquisition point mutation to the resistance of some medicines.
Table 16
| Gene | Disease | Reason | Wild-type sequence (base to be analyzed) restriction endonuclease recognition site and title (needing the inductive base) |
| ?K-ras ?N-ras ?H-ras | Cancer | Codon 12 and 13 point mutation such as k-ras codon 12 are as k-ras codon 13 | GTTGGAGCTGG CCNNNNNNGG?????Bsl?I GGAGTCCTGG CCNNNNNNNGG????Bsl?I |
| ?K-ras | Cancer | The point mutation of codon 12 | GCTGG CCTGG??????????BstNI |
| ?K-ras ?N-ras ?H-ras | Cancer | The point mutation such as the H-ras of codon 61-position 1 | CCAGGAGGAGT CCNNNNNNNGG????Bsl?1 |
| ?H-ras | Cancer | The point mutation of codon 61-position 3 | GGCCGGCCAGG CCNNNNNNNGG????Bsl?1 |
| ?H-ras | Cancer | The point mutation of codon 61 (except the A to T of position 2) | CCAGG CCAGG (CCTGG)????????BstN1 |
| ?H-ras | Bladder cancer | The point mutation of intron D | GTAA TTAA???????????Tru?91 |
| ?HIV-I | The AZT resistance | Point mutation 1. codons 41 2. passwords 70 3. codons 215 | 1.GAAATG ????AATT???????Tsp?509I ??GCAATG???????Bsr?DI 2.AAATGG ??AATT?????????Tsp?509I 3.TTTACC ???TTAA????????Tru?9I |
| The ddI resistance | The point mutation of codon 74 | AAAATTA ??AATT?????????Tsp?509I |
But table 17 is listed in the selection that can have one group of gene of the genetic mutation relevant with disease.The sequence of listing or rule in its lower section for wild-type or for muton and possible sequence variations position illustrates.The analysis of recessive mutation need be distinguished the carrier of heterozygosis and the individuality that isozygotys, and the latter has the risk that disease takes place.For the example below all, the restriction endonuclease of identification wild-type sequence is identified out.Change film conduction gene for cystic fibrosis, the restriction endonuclease of identification mutant nucleotide sequence is also identified.Table 17
| Gene | Disease | Sequence to be analyzed | Sequence type/sequence (base to be analyzed) endonuclease site and title (needing the inductive base) |
| Film conduction regulon is changeed in cystic fibrosis | Cystic fibrosis | Point mutation is positioned at 1. codons, 542 2. codons, 551 3.IVS-4,4. disappearance codons 508 (3bp) | Wild-type sequence 1.ATAGTTCTTGG CCNNNNNNNGG Bsl I CCTGG BstNI 2.CTGAGTGGAGGTCA CCNNNNNNNGG Bsl I GGTCC BsiZI 3.TTATAAGAAGG CCNNNNNNNGG Bsl I 4.AAATATCATCTT GATNNNNATC Bsa BI BsiBI |
| Wild-type sequence 1. codons 542 2. codons 551 3.IVS-4 4. codons 508 | Muton sequence 1.TCTTTGA TTAA Tru 9I 2.GATCAACGAG GATNNNNATC Bsa BI Bsi BI 3.AAGAAGTTAA TTAA Tru 9I 4.AAATATCATTGG CCNNNNNNNGG Bsl I | ||
| α-trypsinase | The hepatic fibrosis wind-puff | Codon 342 point mutation | Wild-type sequence GACCATCGACG CCNNNNNNNGG Bsl I |
| Betaglobulin | β-thalassemia | Point mutation IVS-1 (β °-Mediterranean Sea) | Wild-type sequence CCCTGGGCAGG CCNNNNNNNGG Bsl I |
| Point mutation polyA signal (β+-Black) | Wild-type sequence AATAAA TTAA Tru 9I |
Seldom knew in the past about comprising the effect of heat resistant restriction endonuclease among the PCR.Found in the PCR process, to have simultaneously the selective amplification that restriction endonuclease and dna polymerase activity can cause (i) to contain the inhibition of restriction endonuclease identification/cleavage site sequence amplification and (ii) lack this sequence variant of restriction endonuclease identification/cleavage site, this finds the feasible method of developing REMS-PCR by name, this method can be used for analyzing acquired or genetic polymorphism, comprises point mutation, little disappearance and insertion.When the REMS-PCR method was designed to detect mutant nucleotide sequence, wild-type rather than muton sequence contained the identification/cutting sequence of heat resistant restriction endonuclease.The activity of being limited property of the pcr amplification endonuclease of wild-type sequence suppresses.On the contrary, the muton sequence in the PCR process by the archaeal dna polymerase selective amplification.
The REMS-PCR method also can be designed to the amplification of selectivity mutation inhibiting rather than suppress the amplification of wild-type sequence.If the REMS-PCR method is designed to detect wild-type sequence, muton rather than wild-type sequence contain heat resistant restriction endonuclease identification/cutting sequence.The pcr amplification of muton sequence will being limited property endonuclease activity suppress and wild-type sequence will optionally be increased by PCR.Fail the wild-type sequence of amplifying specific and represent homozygous mutation.Wild-type can be detected and the existence that mutant nucleotide sequence is represented heterozygous mutant can be detected again.
Several REMS-PCR methods are developed the point mutation analysis that is used for K-ras oncogene codon 12.These methods have been utilized Bst NI and the DNA Taq polysaccharase that has enzymic activity simultaneously, or Bst NI and Stoffel fragment polysaccharase, or BslI and DNA Taq polysaccharase.These methods comprise the multi-primers system, comprise a diagnostic primers and a cover or two cover contrast primers.If codon 12 positions 1 and 2 are wild-type, the diagnostic primer is induced identification/cleavage site of BstNI or BslI in the K-ras amplicon.Comprise that in PCR a kind of in these restriction endonuclease into causes the inhibition of wild-type dna profiling amplification and contain the selective amplification of codon 12 positions 1 or 2 mutant DNA templates, therefore with the amplification of these primers can diagnostic code 12 point mutation exist.Extra contrast primer is included in all reactions to confirm reaction conditions, comprises that the amount of template DNA is enough to pass through pcr amplification.These PCR contrast primers can be positioned at the both sides in any zone that does not contain restriction endonuclease identification/cleavage site, and the amplicon that mixes these primers must exist to be used for clearly explaining negative findings.Second contrast primer comprises in the multiplicated system to confirm that restriction endonuclease can mediate the inhibition fully of pcr amplification.The contrast primer that is used for restriction endonuclease must or be induced the identification/cleavage site of the restriction endonuclease that is used for the REMS-PCR method or is positioned at this both sides, site.Lack the amplicon that mixes these primers and clearly indicate positive findings.
The limit that REMS-PCR detects is evaluated by analyze the sample that contains the CaluI DNA (heterozygous mutant of K-ras codon 12) that is diluted among the Sup T1 DNA (K-ras codon 12 wild-types) in the presence of Bst NI and DNA Taq polysaccharase.The detection of diagnostic amplicon shows the existence of the K-ras sequence that codon 12 is.The diagnostic amplicon is containing 1: 10-1: the CaluI of 10,000 ratios with gel electrophoresis and colorimetric analysis: the sample of Sup T1 rather than only contain in the sample of Sup T1 is observed.PCR contrast amplicon is detected at the sample that all comprise Sup T1 DNA.Document shows that the susceptibility of this level will be enough to extract the analysis of DNA from following clinical samples, comprise cutting tissue and vivisection material, cytological sample and body fluid/secretory product such as ight soil, urine and contain the tumour cell that takes off epidermis on a small quantity phlegm (D.Sidranky etc., 1992 science 256,102-1; L.Mao etc., 1994 cancer researches, 54,1634-1637).Application REMS-PCR is proved to the analysis of clinical samples.Detect among the DNA of 2 extractions of the sudden change of K-ras codon 12 in 4 adenocarcinoma of colon but do not have and detect among the DNA that in 4 normal mucous membrane of colon, extracts.
In the extension of REMS-PCR, this method can be finished with following primer, and this primer is induced i simultaneously) exist only in the wild-type sequence restriction endonuclease identification/cleavage site and ii) to the multiple identification/cleavage site of the special restriction endonuclease of all possible mutant nucleotide sequence.With restriction endonuclease to existing of having confirmed divide dividing subsequently of diagnosis amplicon in these amplicons, to suddenly change and evaluation that exact nucleotide under all situations is replaced.
It also is possible developing a kind of like this REMS-PCR system: it causes the selective amplification of mutant nucleotide sequence but does not cause the wild-type sequence amplification to suppress or vice versa.Therefore before analyzing, reactant need be with suitable restriction endonuclease digestion after PCR.This method has the moderate simplicity between the standard REMS-PCR method that the amplification of standard enrichment PCR method and wild-type sequence suppressed fully.
REMS-PCR and multiple catch with detection system be compatible, it can make all method automatization and therefore can be to a large amount of sample real-time analyses.The example of capture systems includes but not limited to i) the PCR primer of the GCN4 bag band GCN4 identification marking of being caught on the flat board; Ii) with avidin or the biotin labeled primer of streptomycete avidin capture; The iii) product of the digoxigenin labeled of catching with anti digoxin antibody; Iv) be attached to the complementary oligonucleotide on latex bead or the magnetic bead.The example of detection system includes, but not limited to i) the biotin labeled PCR primer observed with streptomycete avidin/horseradish peroxidase; Ii) with fluorescein isothiocyanate or the direct mark of alkaline phosphatase molecule; The iii) product of the digoxigenin labeled that detects with anti digoxin antibody.
REMS-PCR provides the sensitivity that is suitable for analyzing the heritable variation relevant with disease and method fast.The ability of keeping restriction endonuclease and dna polymerase activity in the PCR process simultaneously makes the simple method can develop selective amplification varient sequence in reaction, and this is reflected at PCR and just contains all reagent when initial, comprises all enzymes.Thereby reaction can be finished in airtight system and reduce the chance of polluting in the PCR minimizing process.The EMS-PCR method has still less step than other application limitations endonuclease mediation selective amplification and/or the method for analyzing mutant nucleotide sequence.Usually, reaction does not need further operation before detecting, yet, replacing in order to identify definite Nucleotide, this method is not got rid of the subsequent analysis to the diagnosis amplicon.Selective amplification and make REMS-PCR analyze quick, the laborsaving and easier automatization that becomes with the minimizing that restriction endonuclease is analyzed required number of steps.
Those skilled in the art will understand that when not leaving the spirit or scope of the present invention of general description the present invention who shows in can be to specific embodiments does numerous variations and/or improvement.Therefore, the present embodiment is regarded as illustrative and not restrictive in all fields.
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Singh, J., Rao, C.V., Kulkarni N., Simi B. and Reddy B.S. (1994) in colorectal carcinoma chemistry prevention as the molecule marker of transition end points: in the colorectal carcinoma generating process, cause the ras activated to be regulated by sulindac and the different sulfuric acid cyanogen of benzene hexane salt.International cancer is learned magazine 5,1009-1018.
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The patent of quoting
WO 84/01389 (Weinberg etc., Massachusetts engineering college)
EPO?684?315?AI(Becton?Dickinson??and?Company)
US 4683202 (Mullis, K.B.Cetus company)
US 4683195 (Amheim, N. etc., Cetus company)
US 4800159 (Ainheim, N etc., Cetus etc.)
US 4965188 (Ehrlich H.A. etc., Cetus company)
US 5176995 (Erlich H.A. etc., Hoffmann-La Roche company)
Claims (12)
1, a kind of method that detects genetic polymorphism in the individuality, this method may further comprise the steps:
(1) obtains the sample that contains individual nucleic acid;
(2) nucleic acid samples of the method amplification step (1) by comprising thermal cycling and primer, amplification is carried out in the presence of heat-stable restriction endonuclease, it is active that this enzyme keeps in the thermal cycling process, selects primer so that they import the sequence of being discerned by heat-stable restriction endonuclease to amplification from nucleic acid that does not comprise polymorphism or amplification in the nucleic acid of the nucleic acid that comprises polymorphism; With
(3) product of analytical procedure (2) is to detect whether existing of polymorphism.
2, method as claimed in claim 1, the sequence that primer is wherein discerned the heat resistant restriction endonuclease imports amplification from not comprising in the nucleic acid of polymorphism nucleic acid.
3, a kind of method of stopping middle genetic polymorphism that detects, this method comprises following step:
(1) obtains the sample that contains individual nucleic acid;
(2) nucleic acid samples of the method amplification step (1) by comprising thermal cycling and primer, amplification has active heat resistant restriction endonuclease at the same time and exists down and carries out, and the selection of restriction endonuclease is it to be discerned do not comprise the nucleic acid of polymorphism but nonrecognition comprises the nucleic acid of polymorphism or make its identification comprise the nucleic acid of polymorphism but nonrecognition does not comprise the nucleic acid of polymorphism; With
(3) product of analytical procedure (2) is to detect whether existing of polymorphism.
4, method as claimed in claim 3, heat resistant restriction endonuclease identification does not wherein comprise the nucleic acid of polymorphism.
5, as the method for claim 2 or claim 4, wherein this method further comprises following additional step:
(4) with the nucleic acid and the reaction of at least a restriction endonuclease of step (2) amplification, the selection of this at least a restriction endonuclease is to make its digestion comprise the amplification of nucleic acid of specific polymorphism; With
(5) whether detection digests in step (4), and digestion shows the existence of specific polymorphism.
6,, be PCR comprising the method for thermal cycling as the method for arbitrary claim among the claim 1-5.
7, as the method for arbitrary claim among the claim 1-6, wherein the analysis of step (3) comprise detect step (2) amplification of nucleic acid existence whether, the existence whether existence of this amplification of nucleic acid shows polymorphism is whether.
8, as the method for arbitrary claim among the claim 1-7, nucleic acid wherein is DNA.
9, as the method for arbitrary claim among the claim 1-8, heat resistant restriction endonuclease wherein is selected from Bst NI, BslI, Tru 9I and Tsp509I.
10, as the method for arbitrary claim among the claim 1-9, genetic polymorphism wherein detects in one of them of ras proto-oncogene K-ras, N-ras and H-ras or p53 tumor suppressor gene.
11, as the method for claim 10, genetic polymorphism wherein detects in the codon 12 of K-ras.
12, as the method for arbitrary claim among the claim 1-9, genetic polymorphism wherein detects in HIV-I, the conduction of cystic fibrosis commentaries on classics film regulon, alpha antitrypsin or beta-globin.
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| AUPN2452 | 1995-04-13 | ||
| AUPN2452A AUPN245295A0 (en) | 1995-04-13 | 1995-04-13 | Assay for genetic abnormalities |
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| CN1185181A true CN1185181A (en) | 1998-06-17 |
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| CN96194151A Pending CN1185181A (en) | 1995-04-13 | 1996-04-12 | Method and amplifying specific nucleic acid squences |
Country Status (17)
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| US (1) | US6261768B1 (en) |
| EP (1) | EP0871767B1 (en) |
| JP (1) | JPH11503027A (en) |
| KR (1) | KR19980703858A (en) |
| CN (1) | CN1185181A (en) |
| AR (1) | AR001619A1 (en) |
| AT (1) | ATE214429T1 (en) |
| AU (1) | AUPN245295A0 (en) |
| BR (1) | BR9604814A (en) |
| CA (1) | CA2218026A1 (en) |
| DE (1) | DE69619864T2 (en) |
| DK (1) | DK0871767T3 (en) |
| ES (1) | ES2174063T3 (en) |
| IL (1) | IL117884A0 (en) |
| TW (1) | TW546382B (en) |
| WO (1) | WO1996032500A1 (en) |
| ZA (1) | ZA962941B (en) |
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| CN101280338B (en) * | 2007-12-13 | 2011-08-17 | 中国人民解放军第三军医大学第一附属医院 | Nucleic acid amplification method for detecting polymorphism of nucleic acid |
| CN107326066A (en) * | 2004-07-23 | 2017-11-07 | 环太平洋生物技术有限公司 | The urine markers detected for carcinoma of urinary bladder |
| CN114014472A (en) * | 2022-01-07 | 2022-02-08 | 山东玲珑轮胎股份有限公司 | Method and system for treating wastewater generated in waste tire recovery |
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| US6140055A (en) | 1998-03-05 | 2000-10-31 | Johnson & Johnson Research Pty Limited | Zymogenic nucleic acid detection methods and related kits |
| JPH11332562A (en) | 1998-04-28 | 1999-12-07 | Ortho Clinical Diagnostics Inc | Improved methods for extracting nucleic acid from tissue sample and paraffin-embedded tissue |
| US6469159B1 (en) | 1999-04-26 | 2002-10-22 | Robert T. Belly | Methods for extracting nucleic acids from tissue samples and paraffin-embedded tissues |
| ATE278035T1 (en) * | 1999-05-04 | 2004-10-15 | Ortho Clinical Diagnostics Inc | FAST AND EFFECTIVE DNA IMMOBILIZATION FROM A SAMPLE WITHOUT THE USE OF CELL LYSIS REAGENTS |
| AUPQ495700A0 (en) * | 2000-01-05 | 2000-02-03 | Johnson & Johnson Research Pty. Limited | Method for concurrent amplification and real time detection of polymorphic nucleic acid sequences |
| US7262006B1 (en) | 2000-05-01 | 2007-08-28 | Ortho-Clinical Diagnostics, Inc. | Rapid and efficient capture of DNA from sample without using cell lysing reagent |
| CA2424584A1 (en) * | 2000-10-02 | 2002-04-11 | Ortho-Clinical Diagnostics, Inc. | Detection of ras mutations |
| US7052837B2 (en) * | 2001-03-13 | 2006-05-30 | The Board Of Trustees Of The University Of Arkansas | Histoplasma capsulatum catalase sequences and their use in the detection of Histoplamsa capsulatum and histoplasmosis |
| US20030162174A1 (en) * | 2001-06-11 | 2003-08-28 | Sutherland John W. | Detecting nucleic acid deletion sequences |
| KR20040009054A (en) * | 2002-07-22 | 2004-01-31 | 김철욱 | Gene testing method for finding character of each gene of human |
| US20040054160A1 (en) * | 2002-09-16 | 2004-03-18 | Santona Pal | Nucleic-acid ink compositions for arraying onto a solid support |
| DE10315640A1 (en) * | 2003-04-04 | 2004-10-14 | Ignatov, Konstantin | Process for the controlled release of components into a solution |
| WO2004106555A2 (en) * | 2003-05-22 | 2004-12-09 | Dow Agrosciences Llc | High-throughput methods of screening dna for deletions and other mutations |
| US7902345B2 (en) | 2006-12-05 | 2011-03-08 | Sequenom, Inc. | Detection and quantification of biomolecules using mass spectrometry |
| US8652780B2 (en) | 2007-03-26 | 2014-02-18 | Sequenom, Inc. | Restriction endonuclease enhanced polymorphic sequence detection |
| US8999634B2 (en) | 2007-04-27 | 2015-04-07 | Quest Diagnostics Investments Incorporated | Nucleic acid detection combining amplification with fragmentation |
| US9689031B2 (en) | 2007-07-14 | 2017-06-27 | Ionian Technologies, Inc. | Nicking and extension amplification reaction for the exponential amplification of nucleic acids |
| ATE549419T1 (en) | 2007-08-29 | 2012-03-15 | Sequenom Inc | METHODS AND COMPOSITIONS FOR UNIVERSAL SIZE-SPECIFIC POLYMERASE CHAIN REACTION |
| AU2009228312B2 (en) * | 2008-03-26 | 2015-05-21 | Sequenom, Inc. | Restriction endonuclease enhanced polymorphic sequence detection |
| EP2408936A4 (en) * | 2009-03-18 | 2013-01-30 | Sequenom Inc | Use of thermostable endonucleases for generating reporter molecules |
| CA2840542A1 (en) * | 2011-06-28 | 2013-01-03 | Igor Kutyavin | Methods and compositions for enrichment of nucleic acids in mixtures of highly homologous sequences |
| US9352312B2 (en) | 2011-09-23 | 2016-05-31 | Alere Switzerland Gmbh | System and apparatus for reactions |
| JP6009649B2 (en) * | 2013-03-14 | 2016-10-19 | タカラバイオ株式会社 | How to use thermostable mismatch endonuclease |
| US10975415B2 (en) | 2014-09-11 | 2021-04-13 | Takara Bio Inc. | Methods of utilizing thermostable mismatch endonuclease |
| JP6914831B2 (en) * | 2015-03-20 | 2021-08-04 | タカラバイオ株式会社 | High-sensitivity detection method for target nucleic acid |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1284931C (en) | 1986-03-13 | 1991-06-18 | Henry A. Erlich | Process for detecting specific nucleotide variations and genetic polymorphisms present in nucleic acids |
| ATE142273T1 (en) | 1990-06-08 | 1996-09-15 | Behringwerke Ag | METHOD FOR THE QUANTITATIVE DETERMINATION OF DNA SEQUENCES |
| US5455166A (en) * | 1991-01-31 | 1995-10-03 | Becton, Dickinson And Company | Strand displacement amplification |
| AU652214B2 (en) * | 1991-01-31 | 1994-08-18 | Becton Dickinson & Company | Strand displacement amplification "Sealing Clerk - sealing is to be refunded" |
| US5547861A (en) * | 1994-04-18 | 1996-08-20 | Becton, Dickinson And Company | Detection of nucleic acid amplification |
| US5648211A (en) * | 1994-04-18 | 1997-07-15 | Becton, Dickinson And Company | Strand displacement amplification using thermophilic enzymes |
| US5512441A (en) | 1994-11-15 | 1996-04-30 | American Health Foundation | Quantative method for early detection of mutant alleles and diagnostic kits for carrying out the method |
-
1995
- 1995-04-13 AU AUPN2452A patent/AUPN245295A0/en not_active Abandoned
-
1996
- 1996-04-12 CN CN96194151A patent/CN1185181A/en active Pending
- 1996-04-12 AT AT96908918T patent/ATE214429T1/en not_active IP Right Cessation
- 1996-04-12 KR KR1019970707258A patent/KR19980703858A/en not_active Application Discontinuation
- 1996-04-12 EP EP96908918A patent/EP0871767B1/en not_active Expired - Lifetime
- 1996-04-12 US US08/930,828 patent/US6261768B1/en not_active Expired - Fee Related
- 1996-04-12 AR AR33616196A patent/AR001619A1/en unknown
- 1996-04-12 IL IL11788496A patent/IL117884A0/en unknown
- 1996-04-12 ZA ZA962941A patent/ZA962941B/en unknown
- 1996-04-12 BR BR9604814-0A patent/BR9604814A/en not_active Application Discontinuation
- 1996-04-12 CA CA002218026A patent/CA2218026A1/en not_active Abandoned
- 1996-04-12 WO PCT/AU1996/000213 patent/WO1996032500A1/en not_active Application Discontinuation
- 1996-04-12 DE DE69619864T patent/DE69619864T2/en not_active Expired - Fee Related
- 1996-04-12 JP JP8530575A patent/JPH11503027A/en not_active Ceased
- 1996-04-12 ES ES96908918T patent/ES2174063T3/en not_active Expired - Lifetime
- 1996-04-12 DK DK96908918T patent/DK0871767T3/en active
- 1996-04-13 TW TW085104439A patent/TW546382B/en active
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107326066A (en) * | 2004-07-23 | 2017-11-07 | 环太平洋生物技术有限公司 | The urine markers detected for carcinoma of urinary bladder |
| CN101280338B (en) * | 2007-12-13 | 2011-08-17 | 中国人民解放军第三军医大学第一附属医院 | Nucleic acid amplification method for detecting polymorphism of nucleic acid |
| CN114014472A (en) * | 2022-01-07 | 2022-02-08 | 山东玲珑轮胎股份有限公司 | Method and system for treating wastewater generated in waste tire recovery |
| CN114014472B (en) * | 2022-01-07 | 2022-04-26 | 山东玲珑轮胎股份有限公司 | Method and system for treating wastewater generated in waste tire recovery |
Also Published As
| Publication number | Publication date |
|---|---|
| CA2218026A1 (en) | 1996-10-17 |
| IL117884A0 (en) | 1996-08-04 |
| MX9707885A (en) | 1998-06-28 |
| AR001619A1 (en) | 1997-11-26 |
| KR19980703858A (en) | 1998-12-05 |
| DK0871767T3 (en) | 2002-07-08 |
| ATE214429T1 (en) | 2002-03-15 |
| WO1996032500A1 (en) | 1996-10-17 |
| EP0871767A4 (en) | 1999-09-01 |
| JPH11503027A (en) | 1999-03-23 |
| US6261768B1 (en) | 2001-07-17 |
| BR9604814A (en) | 2000-05-09 |
| ZA962941B (en) | 1996-12-04 |
| TW546382B (en) | 2003-08-11 |
| ES2174063T3 (en) | 2002-11-01 |
| EP0871767B1 (en) | 2002-03-13 |
| DE69619864T2 (en) | 2002-11-07 |
| AUPN245295A0 (en) | 1995-05-11 |
| DE69619864D1 (en) | 2002-04-18 |
| EP0871767A1 (en) | 1998-10-21 |
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